Antibodies against phosphorylated VASP ( vasodilator-stimulated phosphoprotein), hybridoma cells for their preparation, and their use

ABSTRACT

The invention relates to antibodies against VASP (vasodilator-stimulated phosphoprotein) which only bind VASP as an antigen when VASP is present in phosphorylated form, to hybridoma cells for their preparation, and to the use of the antibodies or antibody fragments as diagnostic agents and/or therapeutic agents.

[0001] The invention relates to antibodies against VASP(vasodilator-stimulated phosphoprotein) which only bind VASP as antigenwhen VASP is present in phosphorylated form, to hybridoma cells fortheir preparation, and to the use of the antibodies or antibodyfragments as diagnostic agents and/or therapeutic agents.

[0002] Diseases of the vascular system are responsible for a largenumber of chronic and life-threatening diseases, such as cardiacinfarction, stroke, arterial occlusion disease and many forms of kidneyfailure.

[0003] The endothelial cells, which, inter alia, control the bloodcoagulation system, the functional state of the thrombocytes, themigration of inflammatory and tumor cells into the vascular wall, thestate of contraction and growth of smooth muscle cells and consequentlyalso blood pressure and vascular wall structure and alsoneovascularization, are of particular importance for regulating thevascular system. Many of these vascular wall functions are disturbed inserious vascular diseases, a situation which can in the end lead tocardiac infarction, stroke and many forms of kidney failure.

[0004] The endothelium forms the important substances prostacyclin(PGI₂) and nitrogen monoxide (NO), which is also known asendothelium-derived relaxing factor (EDRF), which substances inhibitboth the thrombocytes and the vascular muscle cells. The endothelialfunctions are controlled by said endothelial factors such asprostacyclin (PGI₂) or EDRF, e.g. NO.

[0005] Consequently, in order to treat diseases which are associatedwith an endothelial dysfunction in a specific manner, it is necessary todevelop biochemical parameters which enable an endothelial dysfunctionto be diagnosed and its course to be controlled.

[0006] It is desirable to be able to recognize endothelial dysfunctionsas early as possible, that is at a stage at which irreversible damage,such as atherosclerotic lesions, caused by endothelial dysfunctions hasstill not manifested itself.

[0007] Identification of an endothelial dysfunction at an early stagemakes it possible to develop new therapeutic approaches which can bringabout reversible treatment of the endothelial dysfunction.

[0008] Methods which are known for determining in-vivo endothelialfunctions are invasive detection methods, such as quantitativeangiography, or else non-invasive, image-providing methods.Disadvantages of these methods are that these investigations are carriedout directly on the patient, and are difficult to quantify and veryexpensive.

[0009] It is therefore also desirable to have availablebiochemical/immunobiological methods which make it possible to determineendothelial functions rapidly and simply in biological material ex vivo,for example in cell samples or blood samples by means of routineinvestigations in the laboratory.

[0010] Endothelial functions are those functions which can be regulatedby endothelial factors such as prostacyclin (PGI₂) or EDRF, e.g. NO.

[0011] It is an object of the invention to provide reagents for theevaluation and modulation of endothelial function. According to this andother objects of the invention, an antibody is provided which isdirected to VASP (Vasodilator-stimulated phosphoprotein) and binds VASPonly when VASP is phosphorylated.

[0012] In one aspect of the invention an antibody is disclosed whichbinds VASP only when VASP is phosphorylated at position serine 239(phosphoserine 239 VASP). In another aspect an antibody is providedwhich binds VASP only when VASP is phosphorylated at position serine 157(phosphoserine 157 VASP).

[0013] Different embodiments of the invention include polyclonalantibodies, monoclonal antibodies and antibody fragments. Otherembodiments include a monoclonal antibody produced by the hybridoma cellline 16C2, and particularly Mab 16C2.

[0014] It is a further object of the invention to provide hybridomacells which are useful in manufacturing the antibodies of the invention.Further to this object of the invention hybridoma cell line is providedwhich produces a monoclonal antibody against VASP which binds VASP onlywhen it is phosphorylated. In one embodiment, the hybridoma cell line16C2 (DSM ACC2330) is provided.

[0015] It is yet another object of the invention to provide methods ofevaluating endothelial function. According to this object of theinvention methods are provided for determining the phosphorylationstatus of VASP. In one aspect of the invention biological material iscontacted with an antibody against VASP (vasodilator-stimulatedphosphoprotein), which binds VASP as antigen only when VASP isphosphorylated.

[0016] In another aspect of the invention a quantitative method isprovided which further involves quantifying the amount of VASP antibodywhich binds the biological material. A specific embodiment includes aWestern blotting method, which entails resolving VASP by eletrophoresisand contacting the VASP with an antibody against VASP, which binds VASPonly when it is phosphorylated. Another embodiment is a flow cytometrymethod, which involves contacting a sample with an antibody against VASPwhich binds VASP only when it is phosphorylated.

[0017] In yet another aspect of the invention, methods of diagnosis areprovided. A representative method entails contacting a sample with anantibody against VASP which binds VASP only when it is phosphorylatedand evaluating the phosphorylation state of VASP. In one embodiment,this method involves quantitatively determining the phosphorylation ofVASP in the sample. In another embodiment, methods are described wherethe antibody binds phosphoserine 239 VASP or phosphoserine 157 VASP. Instill another embodiment, the sample is human thrombocytes or humanwhole blood.

[0018] It is yet another object of the invention to provide methods fordetecting markers of endothelial function. In one aspect, a method fordetecting substances which affect the level of cGMP and/or cAMP isprovided which involves testing a sample from patient who has beenexposed to a substance of affecting cGMP and/or cAMP levels, contactingthe sample with an antibody against VASP which binds VASP only when itis phosphorylated and evaluating the phosphorylation state of VASPrelative to a control sample.

[0019] In yet one more aspect, a method is detailed for detectingendothelial dysfunction which involves contacting a sample from apatient with an antibody against VASP which binds VASP only when it isphosphorylated and evaluating the phosphorylation state of VASP relativeto a normal control sample.

[0020] It is a further object of the invention to provide a convenientkit for accomplishing the diagnostic methods disclosed herein. Furtherto this object a diagnostic kit is provided which contains an antibodyagainst VASP which binds VASP only when it is phosphorylated.

[0021] It is still another object of the invention to provide methods oftreating patients suffering from endothelial dysfunction. According tothis and other therapeutic objects of the inventionn a method oftreatment is disclosed where a patient in need of treatment isadministered a therapeutically effective amount of an antibody againstVASP which binds VASP only when it is phophorylated.

[0022] Human VASP (vasodilator-stimulated phosphoprotein), which isphosphorylated in thrombocytes and vascular wall cells in response tohypotensive (vasodilatory) hormones and drugs, has recently beendiscovered, isolated and characterized from the point of view ofmolecular genetics (Haffner et al., EMBO J. 14, 19-27, 1995).

[0023] VASP is an important component of the ANF/NO/cGMP/cGMP proteinkinase signal pathway, which is very important physiologically,pathophysiologically and pharmacologically, and also of the cAMP/cAMPprotein kinase signal pathway (U. Walter, Blick January 1997 WürzburgUniversity, pp. 79-81, 1997). VASP is expressed in almost all human andanimal cells, with particularly high concentrations being found inthrombocytes, vascular smooth muscle cells and fibroblasts. In culturedcells, VASP is associated with focal contacts (cell/matrix contactsites), cell/cell contacts, microfilaments and dynamic membrane regions(e.g. leading edge) (Walter et al., Agents and Actions 45S, 255-268,1995).

[0024] The phosphorylation of VASP in the vascular system correlateswith the inhibition of thrombocyte adhesion/aggregation, the inhibitionof smooth muscle contraction/migration and the inhibition ofparacellular endothelial permeability.

[0025] VASP is phosphorylated and dephosphorylated at three differentsites (serine 157, serine 239 and threonine 278, see Horstrup et al.,Eur. J. Biochem. 225, 21-27 (1994)). Serine 239 is occasionally alsodesignated Serine 238, specifically when the first methionine of VASP isnot included in the count.

[0026] VASP serine 239 is phosphorylated in intact human vascular cells(thrombocytes, endothelial cells and smooth muscle cells) in response tophysiological and pharmacological NO donors and thrombocyte inhibitorsand vasodilators.

[0027] The phosphorylation of VASP serine 239 is mediated, inparticular, by cGMP-dependent protein kinases which are activated, byway of the cGMP, by important hormones such as natriuretic peptides orNO-releasing substances and drugs. Phosphorylation of VASP serine 239 isadditionally mediated by cAMP-dependent protein kinases which areactivated by way of cAMP-increasing hormones and drugs.

[0028] While cAMP-dependent protein kinases principally phosphorylatethe serine 157 position, they also phosphorylate the serine 239 positionof the VASP. VASP phosphorylation at the serine 157 position has alsobeen observed to be closely correlated with inhibition of the binding offibrinogen to glycoprotein IIb-IIIa in human blood platelets (Horstrupet al., Eur. J. Biochem. 225, 21-27 (1994))

[0029] Determination of the degree to which VASP was phosphorylated inbiological material, for example in extracts of tissues and cells, inparticular determination of the phosphorylation of VASP at position 239and/or position 157, would be an important biochemical parameter whosemeasurement would make it possible to develop a diagnostic system fordetecting all cGMP-increasing and/or cAMP-increasing hormones or drugs,such as atrial natriuretic factor (ANF), guanylin, NO-releasingsubstances and drugs, and furthermore enable conclusions to be drawnwith regard to in-vivo endothelial functions.

[0030] Antibodies, that specifically bind to reversibly phosphorylatedproteins have been described in U.S. Pat. No. 5,599,681,WO93/21230 andU. Walter, Blick January 1997 Würzburg University, pp. 79-81, 1997.

[0031] The object of the present invention was to developbiochemical/immunobiological methods which enable the phosphorylationstatus of VASP in biological material to be determined qualitativelyand/or quantitatively in a rapid in simple manner.

[0032] This object was achieved by the provision of antibodies whichonly bind VASP as antigen when the VASP is present in phosphorylatedform.

[0033] The invention consequently relates, in a quite general manner, toantibodies which only bind VASP as antigen when VASP is present inphosphorylated form.

[0034] Within the meaning of the invention, antibodies are to beunderstood as being both polyclonal antibodies and monoclonal antibodies(Mabs) and their fragments, and also SCFV fragments or other syntheticor recombinant protein domains, which specifically recognizephosphorylated regions in the VASP.

[0035] Fragments of antibodies include any portion of the antibody whichis capable of binding the target antigen, in this case VASP or aspecific portion thereof. Antibody fragments specifically includeF(ab′)₂, Fab, Fab′ and Fv fragments. These can be generated from anyclass of antibody, but typically are made from IgG or IgM. They may bemade by conventional recombinant DNA techniques or, using the classicalmethod, by proteolytic digestion with papain or pepsin. See CURRENTPROTOCOLS IN IMMUNOLOGY, chapter 2, Coligan et al., eds., (John Wiley &Sons 1991-92).

[0036] F(ab′)₂ fragments are typically about 110 kDa (IgG) or about 150kDa (IgM) and contain two antigen-binding regions, joined at the hingeby disulfide bond(s). Virtually all, if not all, of the Fc is absent inthese fragments. Fab′ fragments are typically about 55 kDa (IgG) orabout 75 kDa (IdM) and can be formed, for example, by reducing thedisulfide bond(s) of an F(ab′)₂ fragment. The resulting free sulfhydrylgroup(s) may be used to conveniently conjugate Fab′ fragments to othermolecules, such as detection reagents (e.g., enzymes). Fab fragments aremonovalent and usually are about 50 kDa (from any source). Fab fragmentsinclude the light (L) and heavy (H) chain, variable (V_(L) and V_(H),respectively) and constant (C_(L) C_(H), respectively) regions of theantigen-binding portion of the antibody. The H and L portions are linkedby an intramolecular disulfide bridge.

[0037] Fv fragments are typically about 25 kDa (regardless of source)and contain the variable regions of both the light and heavy chains(V_(L) and V_(H), respectively). Usually, the V_(L) and V_(H) chains areheld together only by non-covalent interacts and, thus, they readilydissociate. They do, however, have the advantage of small size and theyretain the same binding properties of the larger Fab fragments.Accordingly, methods have been developed to crosslink the V_(L) andV_(H) chains, using, for example, glutaraldehyde (or other chemicalcrosslinkers), intermolecular disulfide bonds (by incorporation ofcysteines) and peptide linkers. The resulting Fv is now a single chain(i.e., SCFv).

[0038] One preferred method involves the generation of SCFvs byrecombinant methods, which allows the generation of Fvs with newspecificities by mixing and matching variable chains from differentantibody sources. In a typical method, a recombinant vector would beprovided which comprises the appropriate regulatory elements drivingexpression of a cassette region. The cassette region would contain a DNAencloding a peptide linker, with convenient sites at both the 5′ and 3′ends of the linker for generating fusion proteins. The DNA encoding avariable region(s) of interest may be cloned in the vector to fromfusion proteins with the linker, thus generating an SCFv.

[0039] In an exemplary alternative approach, DNAs encoding two Fvs maybe ligated to the DNA encoding the linker, and the resulting tripartitefusion may be ligated directly into a conventional expression vector.The SCFv DNAs generated any of these methods may be expressed inprokaryotic or eukaryotic cells, depending on the vector chosen.

[0040] Preference is given to monoclonal antibodies and their fragmentswhich only bind VASP as antigen when VASP is present in phosphorylatedform.

[0041] Particular preference is furthermore given to monoclonalantibodies and their fragments which only bind VASP, or peptides whichencompass the peptide sequence around serine 239 of VASP, as antigenwhen the serine at position 239 is phosphorylated (phosphoserine 239VASP).

[0042] Within the meaning of the invention, the term VASP is also to beunderstood as meaning derivatives of VASP, i.e. functionally equivalentmoieties, mutants, fragments or variants of VASP, for examplephosphoserine 239 VASP which is additionally phosphorylated at positionserine 157 and/or threonine 278, and also, for example, glycosylationmutants and other covalent modifications and structural elements whichare of importance for protein/protein interaction. Within the meaning ofthe invention, the term VASP encompasses both human VASP and VASP fromother species, in particular from mammals such as rat, mouse, rabbit,dog, pig or monkey.

[0043] Antibodies may be produced by conventional methods for instanceaccording to the technique described by Köhler and Milstein (Köhler andMilstein, Nature 256; 495, 1975). Typically, where selectivity based onphosphorylation state is desired, antibodies are raised against thephosphorylated forms of VASP. The antibodies isolated may be screenedusing conventional methods to ascertain selectivity. Preferably,antibodies are raised against peptide fragments of VASP which containserine 157, serine 239 and/or thereonine 278.

[0044] The length of the antigenic VASP peptide is unimportant, so longas it is capable of eliciting a humoral response. Typical antigenicpeptides are less than about 50 amino acids long. Some preferredpeptides are less than about 20 amino acids in length. Most preferredpeptides are between about 6 and about 12 amino acids long. Preferredpeptides include KLRKVS²³⁹KQ or RKVS²³⁹KQE. The peptide preferably isphosphorylated at serine 157, serine 239 and/or threonine 278. Peptidesmay be obtained by recombinant means. Typically, they are producedeither by proteolytic degradation of VASP or in vitro synthesis andphosphorylation.

[0045] Preference is furthermore given to monoclonal antibodies whichexhibit the abovementioned properties and which can be employed in flowcytometry. This requires the specificity of the novel antibodies to beretained even when the antigen is subjected to conditions which mayalter the conformation of the antigen, as is to be expected in thefixation steps which are customarily employed in flow cytometry. Thenovel antibodies can, for example, be examined for their suitability foruse in flow cytometry simply by means of testing them out.

[0046] The monoclonal antibody 16C2 is particularly preferred.

[0047] The novel antibodies may be prepared using methods which areknown per se to the skilled person. The preparation of hybridoma cellsusing the technique described by Köhler and Milstein (Köhler andMilstein, Nature 256; 495, 1975) may, in particular, be mentioned forpreparing monoclonal antibodies. The specificity of the purifiedantibodies can be checked, for example, using the following testmethods:

[0048] a) Western blotting using recombinant VASP which isphosphorylated by cAMP-dependent or cGMP-dependent protein kinases todiffering extents, in association with which it must only be possible toobserve a positive signal with phosphorylated VASP.

[0049] b) Western blotting using extracts of cells, for example Pkt2cells, which have been transfected with human VASP or with VASP whichhas been mutated in different ways and in which in each case one of thethree phosphorylation sites has been mutated and thereby eliminated(S157A) VASP, (S239A) VASP and (T278A) VASP), and, in each case, humancGMP protein kinase. Those antibodies are suitable in which the positivesignals in the Western blot are eliminated by the S239A mutation but notby the other two mutations.

[0050] c) Western blotting using human thrombocytes which have beentreated with different vasodilators (e.g. prostacyclin or NO donors) oractivators of cAMP protein kinase or cGMP protein kinase. The antibodyis then suitable when a specific band is detected in the extract onlywith phosphorylated VASP. The antibodies can also be screened in ananalogous manner using human fibroblasts and also rat and mousethrombocytes.

[0051] d) Immunofluorescence investigations using fixed humanthrombocytes and endothelial cells. A positive signal is observed whenVASP is present as a phosphoprotein.

[0052] The invention furthermore relates to hybridoma cell lines whichproduce novel monoclonal antibodies, with the hybridoma cell line 16C2,which produces the monoclonal antibody 16C2, being particularlypreferred. The hybridoma cell line 16C2, which produces the monoclonalantibody 16C2, has been deposited in the Deutsche Sammlung vonMikroorganismen und Zellkulturen (German collection of microorganismsand cell cultures), Mascheroder Weg 1b, D-38124 Braunschweig, Germany,in accordance with the rules of the Budapest treaty on Nov. 6, 1997under the following number: DSM ACC2330.

[0053] The novel antibodies are suitable for qualitatively and/orquantitatively, preferably quantitatively, determining both thephosphorylation of VASP which occurs under in-vitro conditions and thatwhich occurs under in-vivo conditions, preferably for determining theserine 239 and/or serine 157 phosphorylation of VASP, particularlypreferably for determining the serine 239 phosphorylation of VASP.

[0054] Quantitative methods of using antibodies are well known in theart and may be found, for example, in CURRENT PROTOCOLS IN IMMUNOLOGY,supra. These methods include enzyme-linked immunosorbant assays (ELISA)radioimmunoassays (RIAs) and the like.

[0055] The novel antibodies are furthermore suitable for qualitativelyand/or quantitatively, preferably quantitatively, determiningphosphorylated VASP, preferably phosphoserine 239 VASP and/orphosphoserine 157 VASP, particularly preferably phosphoserine 239 VASP,in biological material.

[0056] Biological material is, for example, understood as being: cellextracts, tissue extracts, cell slices, cell tissue and cells, such asthrombocytes, leukocytes, endothelial cells, smooth muscle cells andfibroblasts. The biological material can be derived from humans or elsefrom other mammals such as rat, mouse, rabbit, dog, pig or monkey.Biological material of human origin is preferred.

[0057] The novel antibodies can be used to quantitatively determinephosphorylated VASP, in particular phosphoserine 157 VASP and/orphosphoserine 239 VASP, in biological material by means ofquantitatively evaluating autoradiograms of Western blots in accordancewith methods which are known to the skilled person, for example usingNIH gel blotting Image 1.6 software, or else by means ofimmunofluorescence.

[0058] Due to the correlation which exists between the phosphorylationof serine 239 and serine 157 of VASP and the activity of cGMP-dependentand/or cAMP-dependent protein kinases, the novel antibodies are alsosuitable for use as agents for diagnosing cGMP signal pathways and cAMPsignal pathways, in particular for diagnosing cGMP signal pathways.

[0059] The use of the novel antibodies to determine phosphoserine 239VASP in biological material by means of the Western blotting techniqueand immunofluorescence also makes it possible to develop diagnosticmethods for detecting cGMP-increasing substances, hormones or drugs.Examples which may be mentioned are: ANF, guanylin, NO-releasingsubstances or drugs. For example, control of the course and the therapyof NO donors can be monitored, which, inter alia, provides informationon any nitrate tolerance phenomena which may develop.

[0060] Due to the correlation which exists between the phosphorylationof serine 157 of VASP and the activity of cAMP-dependent proteinkinases, the novel antibodies can also be employed as agents fordiagnosing cAMP-increasing substances, hormones or drugs.

[0061] The invention consequently also relates to the use of the novelantibodies or fragments thereof in diagnosis and/or therapy.

[0062] The novel diagnostic methods are methods which can be carried outin the laboratory, outside the human body (ex vivo).

[0063] The novel antibodies can be used for determining the degree ofphosphorylation of VASP in biological material which derives fromdifferent species. Examples which may be mentioned are: man, rat, mouse,rabbit, dog, pig or monkey. Preference is given to using the novelantibodies for determining phosphorylated VASP in biological materialfrom man, rat, mouse or dog. Particular preference is given to using thenovel antibodies for determining phosphorylated VASP in human biologicalmaterial.

[0064] The novel antibodies or fragments thereof can also be used inbiosensors. Biosensors are known per se to the skilled person.Particular preference is given to a method which uses a second specificbinding partner, such as an antibody, a lectin or a receptor. Fordetection and quantification in this case, one of the specific bindingpartners can carry a detectable label. These labels are known per se tothe skilled person and can, for example, be a chromophore, aluminophore, a fluorophore, an enzyme, a radioactive isotope or acolored or colorless particle. Preference is given to a method in whichthe unlabeled, specific binding partner is coupled directly orindirectly, for example by way of another antibody or a biotin/avidinbridge, to a solid phase using methods known per se to the skilledperson.

[0065] The novel antibodies or their fragments can also be radioactivelylabeled by methods known to the skilled person so that they can be usedfor immunoscintigraphy or else for immunotherapy. In addition, thesemonoclonal antibodies can be used as active compound carriers andemployed for the therapy of diseases which are caused by endothelialdysfunctions.

[0066] Following analysis of the complete nucleotide sequence of the Vgenes of Mab 16C2, it is also technically possible to produce antibodyconstructs by, for example, inserting the hypervariable regions into ahuman Mab skeleton (Jones et al., Nature 321, 522-525, 1986; Verhoyen etal., Science 239, 1534-1536, 1988).

[0067] Preference is given to quantifying the antigen-bound antibody inblood platelets by means of flow cytometry, for example carried out onwhole blood samples which have, where appropriate, been fixed usingmethods known to the skilled person. Methods for flow cytometry areknown to the skilled person and can be carried out as described, forexample, in G. Otten and W. M. Yokoyama (1992) Flow cytometry analysisusing the Becton Dickinson FACScan, Current Protocols in Immunology,5.4.1-5.4.19.

[0068] The flow-cytometric analysis of VASP phosphorylation, inparticular VASP serine 239 phosphorylation, using the novel antibodiesis not restricted to human thrombocytes and can also be carried out onother cell types such as lymphocytes, monocytes, leukocytes, endothelialcells and smooth muscle cells.

[0069] The use of flow cytometry to determine the phosphorylation ofVASP, in particular of phosphoserine 239 VASP, in freshly withdrawnhuman thrombocytes with the aid of the novel antibodies makes itpossible to determine ex vivo the in vivo activity of endothelialfactors such as NO and prostacyclin and consequently to assessendothelial function or endothelial dysfunction in cardiovasculardisorders as are found, for example, in arteriosclerosis, hypertension,diabetes, cardiac insufficiency and renal insufficiency.

[0070] Determining VASP phosphorylation, in particular VASP serine 239phosphorylation, with the aid of the novel antibodies also makes itpossible to control the therapy of the abovementioned diseases. Thedevelopment of resistances to therapy, such as nitrate tolerance, canalso be determined.

[0071] The novel antibodies can also be employed as therapeutic agentsin that they affect the phosphorylation status of VASP, and/or itsinteractions with proteins, in biological material.

[0072] The invention consequently also relates to pharmaceuticalpreparations which comprise at least one of the novel antibodies.

[0073] The novel preparations may be used enterally (orally),parenterally (intravenously), rectally or locally (topically). They canbe administered in the form of solutions, powders (tablets or capsulesincluding microcapsules), liposome preparations, lipid complexes,colloidal dispersions, injection solutions or suppositories. Thepharmaceutically customary liquid or solid fillers and extenders,solvents, emulsifiers, glidants, taste corrigents, dyes and/or bufferingsubstances are suitable auxiliary substances for formulations of thisnature. 0.1-100 mg are administered per kg of body weight as anexpedient dose. They are expediently administered in dosage units whichat least comprise the effective daily quantity of the novel antibodies,e.g. 30-3000 mg.

[0074] The daily dose which is to be administered depends on the bodyweight, age, sex and condition of the mammalian subject. However, higheror lower daily doses may also be called for. The daily dose can eitherbe administered by being given on one occasion in the form of a singledosage unit or in the form of several smaller dosage units, or elsebeing given on several occasions, at predetermined intervals, in theform of subdivided doses.

[0075] In order to produce pharmaceutical preparations, the novelantibodies can be worked into therapeutically inert organic andinorganic excipients. Lactose, corn starch or derivatives thereof,tallow and stearic acid or salts thereof are examples of such excipientsfor tablets, coated tablets and hard gelatin capsules. Water, polyols,sucrose, invert sugar and glucose are suitable excipients for preparingsolutions. Water, alcohols, polyols, glycerol and vegetable oils aresuitable excipients for injection solutions. Vegetable oils and hardenedoils, waxes, fats and semisolid polyols are suitable excipients forsuppositories. The pharmaceutical preparations can also comprisepreservatives, solvents, stabilizers, wetting agents, emulsifiers,sweeteners, dyes, flavorings, salts for altering the osmotic pressure,buffers, coating agents, antioxidants and, where appropriate, othertherapeutic active compounds.

[0076] The invention also relates to a process for preparing a novelpharmaceutical, wherein at least one of the novel antibodies is broughtinto a suitable administration form together with a pharmaceuticallysuitable and physiologically tolerated excipient and, where appropriate,other suitable active compounds, additives or auxiliary substances.

[0077] The invention furthermore relates, quite generally, to adiagnostic process for determining the phosphorylation of VASP,preferably the serine 239 phosphorylation of VASP, with the aid of otherspecific probes such as polyclonal antibodies, SCFV fragments or othersynthetic or recombinant protein domains which specifically recognizephosphorylated sequences in VASP, in particular in phosphoserine 239VASP.

[0078] The invention furthermore also relates to a diagnostic processfor determining the serine 157 phosphorylation and/or threonine 278phosphorylation of VASP with the aid of specific probes such asmonoclonal antibodies, polyclonal antibodies, SCFV fragments or othersynthetic or recombinant protein domains which specifically recognizesequences in VASP which contain phosphorylated serine 157 and/orthreonine 278.

[0079] By means of binding to proline-rich peptides, such as zyxin andvinculin, and at the same time binding its own proline-rich amino aciddomain to profilin, VASP makes possible the formation of filamentousactin, with the VASP functioning as an adapter molecule. Disturbance ofthis protein/protein interaction can lead to faulty thrombocyteaggregation or vascular contraction. Thus, the formation of actin/myosinbridges is a prerequisite for the contraction of smooth muscle cells,for example. It has been found that the phosphorylation of VASP at theserine 157 position correlates with inhibition of the fibrinogenreceptor glycoprotein IIb/IIIa (Horstrup et al., Eur. J. Biochem., 225,21-27, 1994). Determination of VASP phosphorylation at the serine 157position consequently makes it possible to search systematically forsubstances or drugs which affect the interaction of VASP with itsintracellular binding partners and are, for example, suitable for thetherapy of cardiovascular diseases which are associated with vasculardamage.

[0080] Particular preference is also given to the embodiments which aredescribed in the implementation examples.

[0081] The following examples serve to clarify the invention and do notrestrict it in any way.

EXAMPLES Example 1

[0082] Isolation of a monoclonal antibody against phosphoserine 239 VASP

[0083] A phosphorylated peptide and a non-phosphorylated peptide, eachof which encompasses the peptide sequence KLRKVS²³⁹ KQ or RKVS²³⁹KQEaround serine 239, are synthesized using an Applied Biosystems peptidesynthesizer (Model 431A) in accordance with the Fmoc chemistry which isfamiliar to the skilled person.

[0084] The phosphoserine in the phosphorylated peptide is incorporatedduring peptide synthesis using Fmoc-serine [PO(Obzl)OH—OH] fromCalbiochem. MS-confirmed peptides are purified using RPC and a VYDAC218TP column (purity>98%).

[0085] After having been activated with bromoacetic acid orbromoacetic-N-hydroxysuccinimide ester (Sigma), the peptides which havebeen prepared in this way are conjugated to thiolated KLH (keyholelimpet hemocyanin, nanoTools). Female Balb/c mice (6 weeks old) areimmunized subcutaneously 4× at 14-day intervals with theKLH-phosphopeptide (10 μg/mouse) containing complete Freund's adjuvantin the first injection and incomplete adjuvant in the 3 followinginjections. The mice are then (2 weeks later) given booster injectionsof 10 μg of immunogen in PBS (phosphate-buffered saline) on threeconsecutive days. 1 day after the last booster injection, the mice aresacrificed and the spleens removed. Spleen cells are isolated and fusedwith non-producing myeloma cells (e.g. PAI-Zellen, J. W. Stocker et al.(1982) Res. Disclosure, 21713) using the established Köhler/Milsteinmethodology.

[0086] A differential screening method usingphosphorylated/non-phosphorylated peptide as well asphosphorylated/non-phosphorylated recombinant human VASP is employed totest hybridoma cells for their ability to secrete antibodies againstphosphoserine 239 VASP.

[0087] For the test using phosphorylated/non-phosphorylated peptide,these peptides are coupled covalently to DNA-BIND-ELISA plates (fromCostar) and the hybridoma supernatents are screened using the ELISAmethod.

[0088] Supernatants which recognize phosphopeptide are additionallyexamined for their ability to recognize completely phosphorylatedrecombinant (E. coli system) human VASP but not the correspondinglydephosphorylated VASP.

[0089] Monoclonal antibodies from the supernatants of the hybridomacells which have been identified by the above-described methods as beingpositive can preferably be purified from serum-free hybridoma cellcultures by means of thiophilic adsorption chromatography (POROS 50-OH,nanoTools).

[0090] Using different test methods, it was possible to identify andcharacterize one of the isolated antibodies (clone 16C2) as being amonoclonal antibody of the mouse IgG1κ class which only recognizes VASPwhen this protein is phosphorylated at the serine 239 position. Antibody16C2 does not recognize other proteins and other VASP phosphorylationsites under the conditions employed.

[0091] Monoclonal antibodies which specifically recognize phosphoserine157 VASP or phosphothreonine 278 VASP can be isolated in analogy withthe above-described method by employing phosphorylated peptides whichencompass the known peptide sequences around serine 157 or threonine 278of the VASP protein, respectively, as the antigen.

[0092] The generation of monoclonal antibodies recognizing VASPphosphorylated at Position Ser 239 is also described in Smolenski etal., J. Biol. Chem., 237, 32, 20029-20035 (1998).

Example 2

[0093] Flow-cytometric analysis of VASP serine 239 phosphorylation

[0094] Analysis of VASP serine 239 phosphorylation in washed humanthrombocytes

[0095] Human thrombocytes are prepared, and VASP phosphorylation isstimulated by incubating with vasoactive substances, as described(Eigenthaler et al., Eur. J. Biochem., 205, 471-481, 1992). The reactionis stopped by fixing, at room temperature for 10 min, with formaldehydeat a final concentration of approx. 3.5%. After a washing procedure(optional), the cells are permeabilized with Triton X-100 and thenwashed. Staining is effected by incubating with the appropriateantibody, which is either directly fluorescence-labeled or colored usinga second antibody which is fluorescence-labeled. Expediently, 0.5-5 μg,preferably 1-2 μg of primary antibody are employed per ml for thispurpose. 1.7 μg/ml are, for example, suitable when Mab 16C2 is used asthe primary antibody. The binding of antibody 16C2 is then determinedand analyzed in the flow cytometer as described, for example, in G.Otten and W. M. Yokoyama (1992) Flow cytometry analysis using the BectonDickinson FACScan, Current Protocols in Immunology, 5.4.1-5.4.19.

Example 3

[0096] Use of Western blotting to determine VASP serine 239phosphorylation in cell extracts

[0097] In parallel with the flow cytometry (Example 2), Western blottingis used to determine the phosphorylation of VASP in cell extracts bymeans of methods which are familiar to the skilled person and which aredescribed, for example, in Eigenthaler et al. (Eur. J. Biochem., 205,471-481, 1992). The concentration of the antibody employed isexpediently between 0.1 and 5, preferably from 0.5 to 1.0, μg/ml,depending on the content of VASP in the sample to be determined. Forexample, for human thrombocytes and rat thrombocytes it is advantageousto use 0.5 μg of antibody/ml, while for human umbilical cord endothelialcells it is advantageous to use 1.0 μg of antibody/ml. In principle,analysis of VASP serine 239 phosphorylation in cell extracts by means ofWestern blot analysis and on fixed cells by means of flow cytometryyielded identical results.

Example 4

[0098] Analysis of VASP serine 239 phosphorylation in whole blood orplatelet-rich plasma (PRP)

[0099] Whole blood or PRP are incubated with vasoactive substances andthe incubation is stopped by fixing, at room temperature for 10 min,with formaldehyde at a final concentration of approx. 3.5%. PRP isprepared from whole blood as described in Eigenthaler et al. (Eur. J.Biochem., 205, 471-481, 1992). Thrombocytes in the PRP are pelleted bycentrifugation and resuspended in physiological buffer (Eigenthaler etal., 1992, see above). The thrombocytes are permeabilized and thenstained as described above for washed thrombocytes.

Example 5

[0100] Time course of the stimulation of VASP serine 239 phosphorylationin washed human thrombocytes following treatment with sodiumnitroprusside (SNP)

[0101] Human thrombocytes were treated with 100 μM SNP. Cell sampleswere withdrawn at times 0, 1, 3 and 5 minutes. The antibody 16C2 wasused to determine the phosphorylation of VASP serine 239 in parallel bymeans of the Western blot analysis of extracts of the cell samples whichhad been withdrawn and by means of flow-cytometric determination carriedout on the human thrombocytes, which had been fixed and permeabilized asdescribed above. The autoradiograms of the Western blots were analyzedquantitatively using NIH gel-blotting image 1.6 software. The increasein antibody 16C2/phosphoserine 239 VASP binding (% increase (5 minvalue=maximum effect=100%) is recorded in Table 1. TABLE 1 ThrombocytesCell extract t (min) % Increase Cytometry Western blotting 0 0 0 1 62.583.5 3 89.3 95.2 5 100.0 100.0

Example 6

[0102] Use of antibody 16C2 to analyze the phosphorylation ofrecombinant VASP by cAMP-dependent protein kinase or cGMP-dependentprotein kinase

[0103] Purified, recombinant hexahistidine-labeled VASP (25 μg/ml) wasphosphorylated by a purified catalytic subunit (11 μg/ml) of cAMPprotein kinase (cAPK) or by purified cGMP protein kinase (cGPK; 24μg/ml). Aliquots were removed from the reaction mixture at the giventimes, mixed immediately with SDS-containing stop solution, boiled andfractionated by means of SDS-PAGE. Phosphorylation of the VASP wasanalyzed by Coomassie Blue staining and by Western blotting using eithera polyclonal VASP antibody (M4, Halbrügge M. et al. J. Biol. Chem. 265,3088-3093 (1990), obtainable from: Alexis Corporation, AlteHauensteinstralβe 4, CH-4448 Läufelfingen, Switzerland) or themonoclonal VASP antibody 16C2. The band below VASP in the Coomassie Bluestaining is cAPK, while that above VASP is cGMP-PK. The SDS-PAGE whichwas performed demonstrated the change in the migratory behavior of VASPfrom 46 to 50 kDA due to the phosphorylation of serine 157, which ispreferred by cAPK. The phosphorylation of serine 239 (detected by the16C2 antibody) is preferred by cGMP-PK.

Example 7

[0104] Analysis of the phosphorylation, in Ptk2 cells, of transfectedVASP containing various phosphorylation mutations

[0105] The phosphorylation of transfected human VASP and VASP mutantswas investigated in Ptk2 cells. Wild-type VASP and VASP mutants each ofwhich contained mutated (inactivated) phosphorylation sites (alterationof serine 157, serine 238 or threonine 277), were transfected into Ptk2cells together with human cGMP protein kinase 1β and expressed under thecontrol of the CMV promoter. Ptk2 cells harbor very little endogenousVASP and cGMP protein kinase. Two days after transfection, the cellswere incubated for 30 min with 30 μM 8pCPT-cGMP and cell extracts werethen isolated. The phosphorylation of VASP in these cell extracts wasthen investigated in Western blots using the polyclonal antibody M4 andthe monoclonal antibody 16C2. A phosphorylation-dependent change of VASPfrom 46 kDa to 50 kDa was no longer present when serine 157 wasinactivated (mutation S157A). A signal recognized by antibody 16C2 wasno longer present when serine 239 was inactivated (S239A). In theseanalyses, mutations of threonine 277 behaved like wild-type VASP.

Example 8

[0106] Analysis of the phosphorylation of VASP in human thrombocytesfollowing stimulation with various vasodilators and cAMP/cGMP analogs

[0107] Washed human thrombocytes (0.7×10⁹ cell/ml) were incubated with 1μM Pg-I₂ (prostacyclin), 0.5 mM 5,6-DCI-cBIMPS (cell membrane-permeablecAMP analog), 10 μM SNP (sodium nitroprusside) or 1 mM 8pCPTcGMP (cellmembrane-permeable cGMP analog). Aliquots (2.8×10⁷ cells) were removedafter the given times, mixed with the SDS stop solution and boiled, andthen investigated for VASP phosphorylation using the Western blottingmethod. The analyses were carried out using the polyclonal antibody M4or the monoclonal antibodies 16C2. Serine 157 phosphorylation wasquantified by the shift of VASP from 46 to 50 kDa, while serine 239phosphorylation was quantified by means of the signal given by the 16C2antibody.

Example 9

[0108] Phosphorylation of VASP in rat thrombocytes

[0109] Rat thrombocytes (0.7×10⁹ cell/ml) were incubated for 5 min with100 μM sodium nitroprusside (SNP), with 10 μM prostaglandin E1 (PgE1) orwithout either of these additions. The extracts of these ratthrombocytes were analyzed by Western blotting. The blot demonstratedthat the phosphorylation-dependent shift of VASP from 46 kDa to 50 kDa(serine 157 phosphorylation) and the phosphorylation-dependent signalgiven by the 16C2 antibody (serine 239 phosphorylation) also took placein rat thrombocytes. Similar results are also obtained using mousethrombocytes.

Example 10

[0110] Immunofluorescence investigations of VASP and phospho-VASPcarried out on human thrombocytes

[0111] Human thrombocytes (in platelet-rich plasma, PRP) were depositedon a glass slide and were allowed to attach and spread for 45 min. Thesethrombocytes were then incubated on the glass slide for 15 min without(A and C) or with 100 μM 8pCPTcGMP (B and D). The cells were thenimmediately fixed with 4% paraformaldehyde and permeabilized with 0.2%Triton X-100. They were then incubated with the polyclonal antibody M4(1:1000) or the monoclonal antibody 16C2, followed by the usualsecondary antibodies. Photographs show the appearance of thephosphorylation-dependent signal when the 16C2 antibody is used, whereasthe signal with the polyclonal M4 antibody (in the immunofluorescence,the sum of 46 kDa and 50 kDa VASP since there is no fractionation) isphosphorylation-independent.

1. An antibody against VASP (vasodilator-stimulated phosphoprotein),which only binds VASP as antigen when VASP is present in phosphorylatedform.
 2. An antibody as claimed in claim 1, which can be used fordetermining the phosphorylation status of VASP in biological material.3. An antibody as claimed in claim 1 and/or 2, which enables thephosphorylation of VASP in biological material to be determinedquantitatively.
 4. An antibody as claimed in one or more of claims 1-3,wherein VASP is only bound as antigen when VASP is phosphorylated atposition serine 239 (phosphoserine 239 VASP).
 5. An antibody as claimedin one or more of claims 1-3, wherein VASP is only bound as antigen whenVASP is phosphorylated at position serine 157 (phosphoserine 157 VASP).6. An antibody as claimed in one or more of claims 1-5, which can beused in the Western blotting technique.
 7. An antibody as claimed in oneor more of claims 1-5, which can be used in flow cytometry.
 8. Anantibody as claimed in one or more of claims 1-7, which is a polyclonalantibody.
 9. An antibody as claimed in one or more of claims 1-7, whichis a monoclonal antibody (Mab) or its fragments.
 10. A monoclonalantibody as claimed in claim 9, which is produced by the hybridoma cellline 16C2.
 11. A monoclonal antibody as claimed in claim 9 and/or 10,which is the Mab 16C2.
 12. A hybridoma cell line, which produces amonoclonal antibody as claimed in one or more of claims 9-11.
 13. Thehybridoma cell line 16C2, which produces the monoclonal antibody 16C2(DSM ACC2330).
 14. The use of an antibody as claimed in one or more ofclaims 1-11 as a therapeutic agent.
 15. The use of an antibody asclaimed in one or more of claims 1-11 as a diagnostic agent.
 16. The useof an antibody as claimed in claim 15 for qualitatively and/orquantitatively determining the phosphorylation of VASP in biologicalmaterial.
 17. The use of an antibody as claimed in claim 15 forqualitatively and/or quantitatively determining phosphoserine 239 VASPor phosphoserine 157 VASP in biological material.
 18. The use of anantibody as claimed in one or more of claims 15-17, wherein thebiological material is human thrombocytes or human whole blood.
 19. Theuse of an antibody as claimed in one or more of claims 15-18 fordetecting substances which affect the level of cGMP and/or cAMP inbiological material.
 20. The use of an antibody as claimed in one ormore of claims 15-19 for determining the in-vivo activity of endothelialfactors.
 21. The use of an antibody as claimed in one or more of claims15-20 for detecting an endothelial dysfunction.
 22. A diagnostic ortherapeutic process, which comprises using polyclonal or monoclonalantibodies, or their fragments, to determine or influence thephosphorylation status of VASP, and/or its protein interactions, inbiological material.
 23. A diagnostic process for qualitatively and/orquantitatively detecting VASP, which is phosphorylated at least at oneof the positions serine 157, serine 239 and threonine 278, in biologicalmaterial, which comprises using antibodies as claimed in one or more ofclaims 1-11.
 24. The diagnostic process as claimed in claim 23, whereinVASP is phosphorylated at position serine 239 (phosphoserine 239 VASP).